Factor A and B of antibiotic A-4696

ABSTRACT

Antibiotic A-4696, produced by Actinoplanes sp., strain ATCC 23342, under submerged aerobic conditions in a liquid culture medium isolated from the fermentation broth by adsorption on activated carbon, eluted therefrom with a 1% sulfuric acid solution in acetone, and purified over sulfuric acid-washed alumina has antibacterial and growth promotant activity.

CROSS-REFERENCE

This application is a divisional of application Ser. No. 678,511, filedApr. 19, 1976, now U.S. Pat. No. 4,064,233, which was acontinuation-in-part of application Ser. No. 533,570, filed Dec. 17,1974, now U.S. Pat. No. 3,952,095, which was a continuation-in-part ofapplication Ser. No. 259,334, filed June 2, 1972, now abandoned, whichwas a continuation-in-part of application Ser. No. 118,674, filed Feb.25, 1971, now abandoned.

BACKGROUND OF THE INVENTION

Tooth decay and gum disease are among the important health problems withwhich man is continuously struggling. The evidence is convincing thatdentobacterial plaques are conductive to tooth caries, or periodontallesions, or both. There is a need for prophylactic programs which willcontrol the formation of dentobacterial plaque or keep such depositsbelow the level at which toxic reactions occur. Antibiotics whichinhibit the growth of plaque-forming microorganisms have been developed,but there remains a need for more effective agents useful in theprevention and treatment of tooth decay and gum disease.

The efficient production of animal proteins for human consumption is acontinuing problem in agriculture. Many agents have been found, amongthem numerous antibiotics, which are effective as feed additives inproducing additional weight gain in growing chickens and swine. There isa never ending need for improved materials which are safe and economicand can be added to animal feed to increase the weight gain and toimprove the feed efficiency. The discovery of agents which willaccomplish this purpose represents a real advance in the art.

SUMMARY

This invention relates to a novel antibiotic and to its preparation.More particularly, this invention relates to the novel nitrogenousantibiotic, arbitrarily denominated herein as A-4696.

The antibiotic of this invention is produced by culturing the organismActinoplanes sp., strain ATCC 23342, in an aqueous nutrient medium undersubmerged aerobic fermentation conditions. The antibiotic is separatedfrom the filtered fermentation broth by adsorption onto an activatedadsorbent and eluted therefrom with an acidic solvent. Antibiotic A-4696is purified as a crystalline compound by adsorption on an acidifiedchromatographic adsorbent and eluted therefrom with an acidic solvent.Preferably, the antibiotic is converted to the hydrochloride or sulfatesalt in an aqueous methanol solution and obtained as a pure crystallinesalt by adding acetone thereto and filtering off the precipitate thatforms. Antibiotic A-4696 possesses antibacterial and growth promotantactivity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The novel antibiotic of this invention is a basic compound capable offorming salts with suitable acids. The characterization data presentedbelow are for antibiotic A-4696 in the form of its hydrochloride salt.The antibiotic is conveniently isolated and characterized as thehydrochloride salt, although other pharmaceutically acceptable salts canbe prepared by employing methods well-known in the art.

Antibiotic A-4696, as the hydrochloride salt, is a white crystallinecompound with a melting point greater that 220° C. It is soluble inwater, and insoluble in solvents such as methanol, acetone, ether,chloroform, pyridine, benzene, aliphatic hydrocarbons, and the like. Itis very stable in solution over a pH range of from about 1.0 to about10.0, at temperatures up to about 27° C.

Electrometric titration of A-4696 hydrochloride in water or indimethylformamide:water (2:1) produces a curve approximating a straightline with a slope of about 0.14 from pH 6.0 to 13.0.

An average of several microanalyses has shown A-4696 hydrochloride tohave approximately the following percent elemental composition: C,51.33; H, 5.79; N, 5.46; O, 30.96; Cl 6.72 percent. The apparentmolecular weight as determined by the vapor pressure osmotic method is1158.

The specific rotation ([α]_(D)), of A-4696 hydrochloride at 25° C. is-42.3° (C=1, H₂ O).

The ultraviolet absorption spectrum of A-4696 hydrochloride in acidicand neutral solutions shows a single absorption maximum at 276 mμ., withan extinction coefficient, E₁ cm.^(1%) of 65.

The infrared absorption spectrum of A-4696 hydrochloride in a mineraloil mull is shown in FIG. 1 of the accompanying drawing. The observeddistinguishable adsorption maxima over the range 2.0 to 15.0 μ. are asfollows: 3.0, 5.8, 5.9, 6.03, 6.15, 6.28, 6.63, 6.85, 7.27, 7.75, 8.1,8.25, 8.9, 9.4, 9.9, 10.1 microns.

The paper chromatographic profile of A-4696 hydrochloride is shown bythe R_(f) values in Table I, below. The values were obtained in eachinstance using Whatman No. 1 paper and the indicated solvent system. Thelocation of the antibiotic on the chromatogram was determined bybioautograph using Bacillus subtilis as the organism.

                  TABLE I                                                         ______________________________________                                        Paper Chromatography of                                                       A-4696 Hydrochloride                                                          Solvent Systems      R.sub.f Value*                                           ______________________________________                                        1                    0.88                                                     2                    0.72                                                     3                    0.80                                                     4                    0.59                                                     5                    0.35                                                     6                    0.77                                                     7                    0.80                                                     8                    0.74                                                     9                    0.87                                                     10                   0.59                                                     11                   0.77                                                     ______________________________________                                         *R.sub.f value is defined as the ratio of the distance traveled by the        antibiotic from the origin to the distance traveled by the solvent front      from the origin.                                                         

Key to Solvent Systems

1. Water saturated with butanol plus 1% p-toluenesulfonic acid.

2. Water saturated with methylisobutyl ketone plus 1% p-toluene-sulfonicacid.

3. Water saturated with methylisobutyl ketone plus 1% p-toluene-sulfonicacid and 1% piperidine.

4. Water:methanol:acetone (12:3:1). The solution is adjusted to pH 10.5with NH₄ OH and then lowered to pH 7.5 with H₃ PO₄.

5. Methanol:0.1N HCl (3:1).

6. One percent methylisobutyl ketone plus 0.5% NH₄ OH in water.

7. Seven percent sodium chloride plus 2.5% methylisobutyl ketone inwater.

8. Ten percent propanol in water.

9. Butanol:ethanol:water (150:15:13.5).

10. Propanol:pyridine:acetic acid:water (15:10:3:12).

11. Water:ethanol:acetic acid (70:24:6).

It is believed that antibiotic A-4696 may be comprised of as many asfive undelineated factors. When a sample of the antibiotic was subjectedto a 16-hour development of a paper chromatographic system utilizing a15:10:3:12 n-butanol, pyridine, acetic acid, water solution as asolvent, five discrete spots were observed. It was not possible toquantify the relative relationship between the spots and no structuraldistinctions could be made.

Two of the afore-mentioned five factors have been isolated and theirphysical properties identified. Factors A and B were separated fromantibiotic A-4696 on a chromatographic column packed with a cross-linkeddextran gel (Sephadex G-25F) set and developed in 1% acetic acid. Eachfactor was monitored by paper chromatography usingn-butanol:pyridine:acetic acid:water (15:10:3:12). The activity waschecked against B. subtilis on a bioautograph.

The integrity of each factor was checked by thin layer chromatographyand the movement of each factor measured and calculated as the fractionof the distance moved by the factor against the front of the solventsystem. The following comparisons were observed:

    ______________________________________                                        Chromatographic                                                                            Solvent     Rf        Rf                                         Medium       System      Factor A  Factor B                                   ______________________________________                                        Cellulose plates                                                                           n-butanol:  0.40      0.51                                                    pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                     Silica Gel plates                                                                          acetone:    0.275     0.525                                      (Quantum Q6F)                                                                              water:                                                                        ammonium                                                                      hydroxide                                                                     (160:40:1)                                                       Whatman #1 paper                                                                           n-butanol:  0.307     0.448                                                   pyridine:                                                                     acetic acid:                                                                  water                                                                         (5:10:3:12)                                                      ______________________________________                                    

The following fermentation medium produced approximately 1000 units/ml.of antibiotic A-4696 in the fermentation broth with factor A accountingfor approximately 90 percent of the antibiotic titer.

    ______________________________________                                        Ingredient            Percent                                                 ______________________________________                                        Glycerol              1.5                                                     Glucose               1.0                                                     Starch, corn          3.5                                                     Yeast extract         2.0                                                     Molasses, beet sugar  1.5                                                     (NH.sub.4).sub.2 SO.sub.4                                                                           0.025                                                   CaCO.sub.3            0.2                                                     K.sub.2 HPO.sub.4     0.05                                                    ______________________________________                                    

Factor B of antibiotic A-4696 was produced in an amount of approximately70-80 percent of the antibiotic titer of 1000 units/ml. of antibioticA-4696 when the fermentation was carried out utilizing the followingfermentation medium:

    ______________________________________                                        Ingredient            Percent                                                 ______________________________________                                        Sucrose               1.0                                                     Peptone               1.5                                                     Soybean meal          0.5                                                     Molasses, beet sugar  1.5                                                     Corn steep liquor     0.5                                                     CaCO.sub.3            0.2                                                     K.sub.2 HPO.sub.4     0.05                                                    MgSO.sub.4 . 7H.sub.2 O                                                                             0.25                                                    ______________________________________                                    

In general, a high ratio of carbohydrate substrates to nitrogensubstrates results in the production of factor A as the major componentof antibiotic A-4696 while a low ratio of carbohydrate substrates tonitrogen substrates in the fermentation medium results in the productionof factor B as the major component of antibiotic A-4696.

When the fermentation media described in Example 5, infra, is employed,approximately equal amounts of factors A and B of antibiotic A-4696 areproduced.

Three additional factors have been separated from antibiotic A-4696 bychromatographic procedures. These factors have been identified asfactors C, D and E. However, it is not known whether these factors areinherent in antibiotic A-4696 or are artifacts of the chromatographicprocesses. Presently, the latter case is believed to be the bestexplanation for the existence of these factors. Factors C, D and E showthe following movements in thin layer chromatographic studies:

    ______________________________________                                                                 Rf      Rf    Rf                                     Chromatographic                                                                            Solvent     Factor  Factor                                                                              Factor                                   Medium     System      C       D     E                                      ______________________________________                                        Cellulose plates                                                                           n-butanol:  0.63    0.69  0.20                                                pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                     Silica Gel plates                                                                          acetone:    0.625   0.725 0.125                                  (Quantum Q6F)                                                                              water:                                                                        ammonium                                                                      hydroxide                                                                     (160:40:1)                                                       Whatman #1 paper                                                                           n-butanol:  0.516   0.602 0.205                                               pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                     ______________________________________                                    

When antibiotic A-4696 was hydrolyzed under mild conditions (0.14N HCl,2 hrs.) an aglycone precipitated and three neutral sugars wereidentified in the supernatant solution. These were identified asglucose, rhamnose, and mannose by thin layer and paper chromatography.

The aglycone was methylated with methyl iodide and oxidized withalkaline permanganate and the resulting extractable organic acids wereesterified with diazomethane. The following structures represent themajor components resulting from the degredation. ##STR1##

These structures are believed to represent most of the aromatic rings inantibiotic A-4696.

When antibiotic A-4696 was reacted with excess benzoylchloride inpyridine and treated with methanolic HCl (reflux),N,O-dibenzoyl-L-ristosaminemethyl glycoside was isolated andcharacterized: ##STR2##

Additionally, a free amino sugar was isolated from antibiotic A-4696.

The fragments detailed above are common to all five factors ofantibiotic A-4696.

Factor A of antibiotic A-4696, as the hydrochloride salt, is a whitecrystalline compound which is soluble in water and insoluble in solventssuch as methanol, acetone, ether, chloroform, pyridine, benzene,aliphatic hydrocarbons and the like. It is very stable in aqueoussolution over a pH range of from about 4 to about 9 at temperatures upto about 27° C.

Microanalysis of factor A hydrochloride of antibiotic A-4696 has shownthe following approximate percent elemental composition: C, 50.21; H,5.48; N, 4.96; O, 30.42; Cl, 6.96 percent.

The specific rotation ([α]_(D)) of factor A hydrochloride of antibioticA-4696 at 25° C. is -81° (C=0.8, H₂ O).

The ultraviolet absorption maximum of factor A hydrochloride ofantibiotic A-4696 in acidic and neutral solutions is at 282 mμ with anextinction coefficient E₁ cm.^(1%) of 35.9.

The infrared absorption spectrum of factor A hydrochloride of antibioticA-4696 in KBr is shown in FIG. 2 of the accompanying drawings. Theobserved distinguishable absorption maxima over the range 2.5 to 16microns are as follows: 3.0 broad, 3.44, 4.25, 4.31, 6.07, 6.20, 6.31,6.65, 7.04, 7.73, 8.10, 8.21, 8.47, 8.89, 9.43, 9.69, 9.83, 10.14,11.07, 11.36, 12.25 microns.

The thin layer chromatographic profile of factor A hydrochloride ofantibiotic A-4696 is shown by the Rf values listed below. The locationof the antibiotic on the chromatogram was determined by bioautographusing B. subtilis as the organism.

    ______________________________________                                        Chromatographic                                                               Medium         Solvent System Rf Value                                        ______________________________________                                        Cellulose plates                                                                             n-butanol:     0.40                                                           pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                   Silica gel plates                                                                            acetone:       0.275                                           (Quantum Q6F)  water:                                                                        ammonium                                                                      hydroxide                                                                     (160:40:1)                                                     Whatman #1 paper                                                                             n-butanol:     0.307                                                          pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                   ______________________________________                                    

Factor B of antibiotic A-4696, as the hydrochloride salt, is a whitecrystalline compound which is soluble in water and insoluble in solventssuch as methanol, acetone, ether, chloroform, pyridine, benzene,aliphatic hydrocarbons and the like. It is very stable in aqueoussolution over a pH range of from about 4 to about 9 at temperatures upto about 27° C.

The average microanalysis of factor B hydrochloride of antibiotic A-4696shows the following approximate percent elemental composition: C, 50.12;H, 4.98; N, 5.51; O, 29.45; Cl, 6.30 percent.

The specific rotation ([α]_(D)) of factor B hydrochloride of antibioticA-4696 at 25° C. is -108.3 (C=1, H₂ O).

The ultraviolet absorption maximum of factor B hydrochloride ofantibiotic A-4696 in acidic and neutral solutions is at 280 mμ with anextinction coefficient E₁ cm.^(1%) of 51.2.

The infrared absorption spectrum of factor B hydrochloride of antibioticA-4696 in KBr is shown in FIG. 3 of the accompanying drawings. Theobserved distinguishable absorption maxima over the range of 2.5 to 16microns are as follows: 3.0 broad, 3.44, 4.25, 4.31, 6.07, 6.20, 6.31,6.66, 7.04, 7.75, 8.13, 8.24, 8.48, 8.91, 9.43, 9.69, 9.83, 10.14,11.07, 11.36, 12.25 microns.

The thin layer chromatographic profile of factor B hydrochloride ofantibiotic A-4696 is shown by the Rf values listed below. The locationof the antibiotic on the chromatogram was determined by bioautographusing B. subtilis as the organism.

    ______________________________________                                        Chromatographic                                                               Medium         Solvent System Rf Value                                        ______________________________________                                        Cellulose plates                                                                             n-butanol:     0.51                                                           pyridine:                                                                     acetic acid:                                                                  water                                                          (15:10:3:12)                                                                  Silica gel plates                                                                            acetone        0.525                                           (Quantum Q6F)  water                                                                         ammonium                                                                      hydroxide                                                                     (160:40:1)                                                     Whatman #1 paper                                                                             n-butanol:     0.448                                                          pyridine:                                                                     acetic acid:                                                                  water                                                                         (15:10:3:12)                                                   ______________________________________                                    

The antibiotic activity of both factors A and B hydrochlorides ofantibiotic A-4696 has been established as being substantially the sameas that exhibited by antibiotic A-4696 against B. subtilis.

By employing methods well-known in the art, pharmaceutically acceptablesalts of antibiotic A-4696, and factors A and B thereof can be preparedwith mineral acids such as hydrochloric, hydrobromic, sulfuric,phosphoric, and the like, and also with organic acids such as citric,tartaric, maleic, p-toluenesulfonic, salicylic, fumaric, acetic,propionic, and the like. The antibiotic salts of such acids can beprepared, for example, by acidifying a solution of the antibioticfree-base with the desired acid and precipitating the salt thusly formedby introducing ten volumes of acetone to the solution containing theA-4696 acid salt. The salts can likewise be prepared in certaininstances by ion exchange on an ion exchange column. Other commonly usedmethods for the preparation of antibiotic salts can also be employed.

The novel antibiotic of this invention has an inhibiting action againstthe growth of many microbial organisms which are pathogenic to man,animals and plant-life, and is, therefore, useful in suppressing thegrowth of such organisms. The levels at which A-4696 hydrochloride showsinhibition against the growth of the illustrative organisms are setforth numerically in Table II, below. The inhibition levels weredetermined by either the agar-dilution test or the broth-dilution test,and are stated in terms of the minimum inhibitory concentration (MIC),micrograms (s) per milliliter (mcg./ml.). (Identified in Table II by theletter "ad" and "bd", respectively).

In the agar-dilution test the test organism is streaked or implanted onagar plates containing various concentrations of A-4696 hydrochloride inthe agar. The test plates are incubated at 37° C. for 48 hours, and theMIC is determined as the plate at the lowest concentration of theantibiotic where growth of the test organism is inhibited.

In the broth-dilution test a series of tubes containing nutrient brothand varying concentrations of A-4696 hydrochloride are inoculated withthe test organism and incubated at 37° C. for 24 hours. The MIC isdetermined as the lowest antibiotic concentration where no growth ispresent in the tube.

                  TABLE II                                                        ______________________________________                                                             Minimum Inhibitory                                                            Concentration                                            Test Organism        (mcg./ml.)                                               ______________________________________                                        Staphylococcus aureus 3055                                                                         12.5      ad                                                                  6.25      bd                                             Bacillus subtilis    0.78      ad                                             Mycobacterium avium  0.4       ad                                             Streptococcus faecalis                                                                             3.12      ad                                             Trichophyton mentagrophytes                                                                        0.2       ad                                             Vibrio coli          12.5      bd                                             Mycoplasma gallisepticum                                                                           50.0      bd                                             Staphylococcus aureus                                                                              10.0      bd                                             (penicillin resistant                                                         Staphylococcus aureus (methicillin                                                                 5.0       bd                                             resistant                                                                     Diplococcus pneunoniae                                                                             3.12      bd                                             Clostridium perfringens                                                                            1.25      bd                                             Clostridium tetani   2.5       bd                                             Corynebacterium gravis                                                                             1.25      bd                                             Lactobacillus casei  >100      ad                                             Leuconostoc citrovorum                                                                             >100      ad                                             Escherichia coli     >100      ad                                             Proteus sp.          >100      ad                                             Pseudomonas sp.      >100      ad                                             Salmonella sp.       >100      ad                                             Vibrio metschnikovii >100      ad                                             Saccharomyces pastorianus                                                                          >100      ad                                             Candida albicans     >100      ad                                             ______________________________________                                    

Antibiotic A-4696 hydrochloride when given by subcutaneous injection tomice has in vivo antimicrobial activity against infectious organisms;for example: the ED₅₀ values (effective dose to protect 50% of the testanimals) in illustrative infections are as follows when two doses areemployed: Staphylococcus aureus, 9.2 mg./kg.; Streptococcus pyogenes,0.68 mg./kg.; and Diplococcus pneumoniae, 0.76 mg./kg.

The novel antibiotic of this invention is also effective in inhibitingthe growth of microorganisms which contribute to the development ofperiodontal disease and tooth decay. For example, a solution of A-4696hydrochloride exhibits antimicrobial activity against plaque formingorganisms as illustrated by the following test system: Tubes of nutrientbroth containing 5% sucrose are inoculated with a cariogenicmicroorganism. Glass rods are immersed in the medium and the tubes areincubated at 37° C. overnight after which the layer of plaque (primarilycells and dextran) forms on the surface of the rods. The rods are thentransferred to solutions containing varying concentrations of A-4696hydrochloride and allowed to remain in contact with the antibiotic for5, 10, and 15 minutes. After the appropriate time has elapsed, the rodsare rinsed with sterile, deionized water, and immersed in uninoculatedmedium containing sucrose and bromthymol blue. After incubatingovernight at 37° C., growth is detected by observing the color changefrom green to yellow due to the acid production of the organisms whengrown in a sucrose containing medium.

A solution of A-4696 hydrochloride at a concentration of 0.01% waseffective against an unidentified cariogenic Streptococcus sp. when incontact with the plaque encrusted rods for ten minutes. The growth ofthis organism was prevented by A-4696 hydrochloride at a 0.1%concentration when the solution was in contact with the test rod for 5minutes.

Moreover, A-4696 hydrochloride inhibits the growth of the plaque-formingorganism Odontomyces viscosus at a concentration of 0.25 mcg./ml. in abroth-dilution test.

The incorporation of A-4696 or one of its acid addition salts into anappropriate toothpaste, gel, powder, or the like, or a suitablemouthwash, or other oral hygiene preparation, can provide an effectivemethod for inhibiting the development of dental caries and periodontaldisease. Alternatively, a solution of A-4696, or one of its acidaddition salts at an appropriate concentration can be applied to thesurface of the gums and teeth with a suitable swab.

Also, it has been found that the daily oral administration of a growthpromoting quantity of antibiotic A-4696, or a suitable derivativethereof, as a component in the feed consumed by poultry and swinesignificantly accelerates the growth rate of the animals and improvesthe efficiency of feed utilization. The daily ingestion by poultry andswine of antibiotic A-4696 or a suitable derivative thereof in an amountof from about 0.5 mg. to about 25 mg./kg. of body weight results in afaster growth than that registered by animals fed the same basal rationwithout the active agent. For example, broiler cockerels ingesting about2 mg./kg. of antibiotic A-4696 hydrochloride daily gained 4.35 percentmore weight and consumed 3.29 percent less total feed from age 7 days toage 28 days than comparable cockerels eating the same basal ration. Theterm "basal ration" as used herein refers to the total feed intake ofthe animal, which may take the form of a complete feed ration into whichare incorporated in one composition all of the elements constituting thedietary requirements of the animal, or may be regarded as the sum of allof the elements contained in various feedstuffs, concentrates,supplements, mineral, vitamin or medicated premixes, roughages, or thelike, which are fed to the animal. The composition and analysis of atypical poultry basal ration fed as a complete feed is shown in TableIII, below.

                  TABLE III                                                       ______________________________________                                        Broiler Basal Ration                                                          Ingredients          Percent   Lbs./Ton                                       ______________________________________                                        Corn, Yellow, Ground 53.90     1078                                           Soybean Oil Meal Sol. Ext. 50%                                                                     29.00     580                                            Distillers Dried Sol. (Corn)                                                                       2.50      50                                             Alfalfa Meal, Dehydrated 17%                                                                       2.50      50                                             Whey, Dried          1.00      20                                             Fish Meal (Menh) + Sol.                                                                            4.00      80                                             Animal Fat           4.00      80                                             Dicalcium Phosphate, Feed Grade                                                                    1.70      34                                             Calcium Carbonate    .50       10                                             Salt (NaCl)          .30        6                                             Trace Mineral Premix.sup.1                                                                         .10        2                                             Vitamin Mix CK-O1 (1.02).sup.2                                                                     .50       10                                             Methionine Hydroxy Analogue (80%)                                                                  .062      1.25                                           Totals               100.062   2001.25                                        ______________________________________                                         .sup.1 Trace Mineral Premix contains: 6.7% manganese as manganese sulfate     0.09% iodine as potassium iodide, 0.17% copper as copper oxide, 7.5% zinc     and zinc carbonate, and 1.7% iron as ferrous sulfate.                         .sup.2 Each pound contains: 450,000 I.U. vitamin A, 120,000 I.C.U. vitami     D.sub.3, 1,000 I.U. vitamin E, 400 mg. riboflavin, 3,600 mg. niacin, 966      mg. d-panthothenic acid, 26,037 mg. choline, 1 mg. vitamin B.sub.12 and       100 mg. menadione sodium bisulfite.                                      

Moreover, pigs ingesting from about 1.25 to about 6 mg./kg./dayantibiotic A-4696 hydrochloride gained about 5.7 percent more weight andconsumed about 4.1 percent less feed per pound of weight gained from thetime they weighted about 50 pounds until they reached slaughter weightat about 200 pounds than comparable pigs eating the same basal rationbut without the active compound. The composition and analysis of atypical swine basal ration is shown in Table IV, below.

                  TABLE IV                                                        ______________________________________                                        Swine Basal Starter Ration                                                    Ingredients          Percent   Lbs./Ton                                       ______________________________________                                        Corn, Yellow, Ground 69.25     1385                                           Alfalfa Meal, Dehydrated 17%                                                                       2.50      50                                             Soybean Oil Meal, Sovent Extracted                                                                 17.00     340                                            Dehulled 50%                                                                  Meat Scraps,55%      2.50      50                                             Fish Meal with Solubles                                                                            2.50      50                                             Distillers Dried Solubles (Corn)                                                                   2.50      50                                             Animal Fat           2.00      40                                             Salt (NaCl)          0.50      10                                             Dicalcium Phosphate, Feed Grade                                                                    0.50      10                                             Calcium Carbonate    0.20       4                                             Trace Mineral Premix.sup.1                                                                         0.05       1                                             Vitamin Premix - SW-03.sup.2                                                                       0.05      10                                             Totals               100.00    2000                                           ______________________________________                                         .sup.1 Trace Mineral Premix contains: 10.00% manganese as manganese           sulfate, 0.30% iodine as potassium iodide, 0.10% cobalt as cobalt             carbonate, 6.00% iron as ferrous carbonate, 1.00% copper as copper oxide,     10.00% zinc as zinc oxide, and 11.50% maximum and 8.50% minimum calcium       carbonate.                                                                    .sup.2 Each pound contains: 35,000 USP units vitamin D.sub.2, 200 mg.         riboflavin, 1,000 mg. niacin, 735 mg. pantothenic acid, 8,700 mg. choline     and 2 mg. vitamin B.sub.12.                                              

In a preferred embodiment of the present invention, antibiotic A-4696,or a suitable derivative thereof, is administered orally in a suitablefeed in which the active compound is present in an amount of from about2 to about 200 grams per ton of total feed, the exact concentrationemployed being that which is required to provide for the active agentwhen normal amounts of feed are consumed. The addition of the activecompounds of this useful process of this invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining from about 1 to about 100 grams of antibiotic A-4696, or asuitable derivative thereof, per pound of premix and incorporating thepremix into the complete ration. Alternatively, an intermediateconcentrate or feed supplement containing the active agent can beblended into the feed.

The preparation of an appropriate feed premix can be effected bygrinding the active compounds utilized in the novel process of thisinvention to a powder and admixing with a suitable premix carrier. Theterm "suitable premix carrier" as used in this disclosure may refer toan edible feedstuff which constitutes a normal dietary ingredient of theanimal, such as, alfalfa grits, solvent-extracted soybean feed, groundcorn, and the like, or a combination of components of a feed ration, ora physiologically utilizable mineral or vitamin concentrate orsupplement, or the like. Alternatively the term may refer to a blandnon-irritating material which is acceptable by the animal, but which isnot physiologically utilizable, as for example, ground corn cobs,exfoliated hydrobiotite, or the like. The premix so prepared is thenadmixed with whatever feed ration is being fed to the animals at thetime of administering the active agents employed in this useful processof the present invention. The feed premix can be diluted first with afeed supplement or feed concentrate to a desired concentration of theactive compound, and the medicated supplement or concentrate can eitherbe fed concurrently with the remainder of the ration or mixed into thefinal feed.

An alternative procedure for preparing the premix comprises dispersingthe active compound in a suitable vehicle such as an edible vegetableoil, an edible glyceride, or an edible glycol, and spraying suchdispersion onto the premix carrier with suitable mixing.

This aspect of the present invention is further illustrated by thefollowing examples.

EXAMPLE 1

This test was run to compare weight gains and feed efficiencies in youngbroiler chickens when antibiotic A-4696 hydrochloride was fed in anamount of 45.4 grams of active compound per ton of basal ration withthose observed when the basal ration with no growth promotant includedtherein was employed.

Sixty battery pens of ten chicks each were utilized. The birds were oneto five-day old broiler chicks (Arbor Acres Strain 50) at the start ofthe test. All of the birds were provided with the same basal ration(Table III, above) for the duration of the test. Thirty pens, a total of300 birds, were provided with the basal ration without a growthpromotant. Thirty pens, a total of 300 birds, were provided with thesame ration containing, in addition, antibiotic A-4696 hydrochloride ata level of 45.4 grams per ton. The chicks were randomly assigned to thepens, and the test was conducted in a climatecontrolled environment foreither 10 or 17-day periods. Continuous lights and ad libitum feedingwere used throughout the trials.

All birds were group weighed at the beginning and end of the trials.Average weight gains and feed efficiency were calculated for the periodof the test.

The birds fed antibiotic A-4696 for 17 days produced a 6.06 percentgreater weight gain and showed a 6.45 percent improvement in feedefficiency over the birds receiving no growth promotant.

Table V shows the test results.

                                      TABLE V                                     __________________________________________________________________________    Weight Gains and Feed Efficiency in Young Chicks                              Fed A-4696 Hydrochloride in the Feed Ration.                                                                                     Feed Conversion                        A-4696 Hydrochloride                                                                      No. of Days                                                                           No. of Birds in                                                                         Average Gain,                                                                          Efficiency                 Feed Ration g./ton of Feed                                                                            on Treatment                                                                          Treatment g./Bird  LB. Feed/LB.               __________________________________________________________________________                                                       Gain                       Basal ration                                                                              0           10      40        147      1.53                       Basal ration plus                                                                         45.4        10      40        146      1.47                       A-4696 Hydro-                                                                 chloride                                                                      Basal ration                                                                              0           10      20        146      1.50                       Basal ration plus                                                                         45.4        10      20        153      1.45                       A-4696 Hydro-                                                                 chloride                                                                      Basal ration                                                                              0           17      80        295      1.56                       Basal ration plus                                                                         45.4        17      80        320      1.46                       A-4696 Hydro-                                                                 chloride                                                                      Basal ration                                                                              0           17      80        300      1.51                       Basal ration plus                                                                         45.4        17      80        318      1.43                       A-4696 Hydro-                                                                 chloride                                                                      Basal ration                                                                              0           17      80        287      1.58                       Basal ration plus                                                                         45.4        17      80        307      1.47                       A-4696 Hydro-                                                                 chloride                                                                      __________________________________________________________________________

EXAMPLE 2

This test was run to compare weight gains and feed efficiencies whenantibiotic A-4696 hydrochloride was fed in varying amounts and fordifferent periods of time. Battery trials were run at concentrations of1,5,10, and 20 grams of active compound per ton of basal ration for 21days (7-28 days of age). Floor-pen trials were run at concentrations of5 and 20 grams of active compound per ton of basal ration for 28 and 56days (7-35 and 7-63 days of age, respectively).

The battery trials were run with 8 birds in each pen. The floor-pentrials had 50 birds in each pen. All of the birds were in housed in atemperature controlled environment and provided with ad libitum feedingand continuous lights. All of the chicks were fed the same chick starterration (Table III, above) during the first 21 days of the test, and, inthe case of the floor-pen trials, the same finisher ration (Table VI,below) during the remainder of the test.

The birds in the 21-day battery trial showed statistically significantimprovement in weight gain at the 5, 10, and 20 g. ton level. There werestatistically significant improvements in weight gain and feedefficiency in the chickens in the 28-day floor-pen trial at the 20g./ton level.

The results of the test are shown in Table VII.

                  TABLE VI                                                        ______________________________________                                        Broiler Finisher Ration                                                       Ingredients          Percent   Lbs./Ton                                       ______________________________________                                        Corn, Yellow, Ground 65.8      1316                                           Soybean Oil Meal Sol. Ext. (50%)                                                                   26.4       528                                           Animal Fat           3.90      78.0                                           Dicalcium Phosphate, Feed Grade                                                                    2.30      46.0                                           Calcium Carbonate    .70       14.0                                           Salt (NaCl)          .40       8.0                                            Trace Mineral Premix.sup.1                                                                         .10       2.0                                            Vitamin Mix CK-01 (1.02).sup.2                                                                     .50       10.0                                           Totals               100.10    2002.0                                         ______________________________________                                         .sup.1 Trace Mineral Premix contains: 6.7% manganese as manganese sulfate     0.09% iodine as potassium iodide, 0.17% copper as copper oxide, 7.5% zinc     and zinc carbonate, and 1.7% iron as ferrous sulfate.                         .sup.2 Each pound contains: 450,000 I.U. vitamin A, 120,000 I.C.U. vitami     D.sub.3, 1,000 I.U. vitamin E, 400 mg. riboflavin, 3,600 mg. niacin, 966      mg. d-panthothenic acid, 26,037 mg. choline, 1 mg. vitamin B.sub.12 and       100 mg. menadione sodium bisulfite.                                      

                                      TABLE VII                                   __________________________________________________________________________    Weight Gains and Feed Efficiency in Young Chickens                            Fed A-4696 Hydrochloride in the Feed Ration                                                       No.     No.                                                           A-4696  of Days of Chickens                                                                           Average                                                                              %    Feed Conversion                                                                         %                   Type of                                                                            Feed   Hydrochloride                                                                         on      in      Gain   Improve-                                                                           Efficiency                                                                              Improve-            Trial                                                                              Ration g./ton of Feed                                                                        treatment                                                                             Treatment                                                                             g./Chicken                                                                           ment Feed/lb.                                                                                ment                __________________________________________________________________________    Battery                                                                            Basal  0       21      656     537.5  --   1.694     --                       Ration                                                                   "    Basal                                                                         Ration plus                                                                   A-4696 Hy-                                                                    drochloride                                                                          1       21      192     536.5  -0.19                                                                              1.686     0.47                "    "      5       21      192     553.0  2.88 1.639*    3.36                "    "      10      21      192     5559.1*                                                                              4.02 1.648*    2.79                "    "      20      21      384     560.9* 4.35 1.640*    3.29                Floor-                                                                             Basal  o       28      900     550.7  --   1.794     --                  Pen  Ration                                                                   "    Basal                                                                         Ration plus                                                                   A-4696 Hy-                                                                    drochloride                                                                          5       28      300     570.3  3.56 1.727     3.88                "    "      20      28      900     582.9* 5.85 1.704*    5.28                Floor-                                                                             Basal                                                                    Pen  Ration 0       56      300     1774   --   2.22      --                  "    Basal                                                                         Ration                                                                        Plus                                                                          A-4696 Hy-                                                                    drochlo-                                                                      ride   5       56      300     1810   1.02 2.17      2.30                "    "      20      56      300     1822   2.70 2.16      2.77                __________________________________________________________________________     *Statistically significant, P< 0.05                                      

EXAMPLE 3

This test was run to determine the effect of feeding antibiotic A-4696hydrochloride to weanling pigs and its influence on the average dailyweight gain and feed efficiency was tested.

Forty-four weanling pigs at 4 to 5 weeks of age were weighed andassigned to 4 experimental groups of 11 pigs each on the basis of weightand litter. Pigs were housed in enclosed buildings with adequate heatand ventilation, and feed and water were available ad libitum at alltimes. In each test the pigs were divided into two groups, one of thegroups received the basal ration (Table IV, above) with no antibioticadded, and the other group received the same basal ration plusantibiotic A-4696 hydrochloride. Table VIII, shown below, lists theimprovement in average daily gain and feed efficiency in those pigswhich were fed the A-4696 hydrochloride as contrasted with those whichreceived only the basal ration. The pigs getting feed containing 50g./ton of A-4696 hydrochloride gained 11.8 percent more weight and had a10.0 percent lower feed consumption than the control animals receivingno antibiotic.

                                      TABLE VIII                                  __________________________________________________________________________    Average Daily Weight Gain and Feed Efficiency in Weanling Pigs                Fed Antibiotic A-4696 Hydrochloride as a Part of the Daily Diet                                                              Feed Conversion                          A-4696 Hydrochloride                                                                      No. of Pigs                                                                           No. of Days                                                                           Average Daily                                                                          Efficiency                     Feed Ration                                                                             g./ton      in Test on Test Gain Pounds                                                                            LB. Feed/LB.                   __________________________________________________________________________                                                   G                              Basal ration                                                                            0           11      28      0.67     1.91                           Basal ration plus                                                                       20          11      28      0.95     1.77                           A-4696 Hydro-                                                                 Chloride                                                                      Basal ration                                                                            0           11      34      1.10     2.41                           Basal ration plus                                                                       50          11      34      1.23     2.17                           A-4696 Hydro-                                                                 Chloride                                                                      __________________________________________________________________________

EXAMPLE 4

This test was run to determine the effect of feeding antibiotic A-4696hydrochloride to pigs from the time they are about 50 pounds each untilthey reach slaughter weight, (about 200 pounds).

Four groups of pigs, each numbering 72 animals, were housed in enclosedbuildings with adequate heat and ventilation. Feed and water wereavailable ad libitum. All pigs were fed a basal ration (Table IX, below)for two weeks before initiating the trials. One group of 72 pigsreceived no antibiotic for the duration of the test, and the other threegroups were fed antibiotic A-4696 hydrochloride at levels of 4.54, 18.1and 45.4 g./ton of feed. All animals received the same basal ration atall times.

The pigs fed antibiotic A-4696 hydrochloride at the 45.4 g./ton levelgained 5.7 percent more weight and consumed 4.1 percent less feed thanthe control animals receiving no antibiotics.

The pertinent data generated in this test are nummarized in Table X,below.

                  TABLE IX                                                        ______________________________________                                        SWINE GROWER RATION                                                           Ingredient            Percent  Lbs./Ton                                       ______________________________________                                        Corn, Yellow, Ground  73.2     1464                                           Alfalfa Meal, Dehydrated, 17%                                                                       2.5      50                                             Soybean Oil Meal, Solvent Extracted                                           Dehulled, 50%         12.6      246                                           Meat Scraps, 55%      2.5      50                                             Fish Meal with Solubles                                                                             2.5      50                                             Distillers Dried Solubles (Corn)                                                                    2.5      50                                             Animal Fat            2.0      40                                             Calcium Carbonate     0.7      14                                             Dicalcium Phosphate, Feed Grade                                                                     0.5      10                                             Salt (NaCl)           0.5      10                                             Trace Mineral Premix, AN-03.sup. 1                                                                  0.1       2                                             Swine Vitamin Premix, SN-03.sup. 2                                                                  0.5      10                                             Methionine Hydroxy Analogue, 90%                                                                    0.2       4                                             Total                 100.3    2000                                           ______________________________________                                         .sup.1 Each kg. of premix contains the following: 50 gm of manganese          sulfate, 100 gm. of zinc as zinc carbonate, 50 gm. of iron as ferrous         sulfate, 5 gm. of copper as copper oxide, 1.5 gm. iodine as potassium         iodide; and 150 gm. maximum and 130 gm. minimum calcium as calcium            carbonate.                                                                    .sup.2 Each kg. of premix contains the following: 77,161 USP units vitami     D.sub.2 ; 440.0 mg. riboflavin; 2,240.6 mg. niacin; 1,602.4 mg.               pantothenic acid; 19,180 mg. chlorine; 4.4 mg. vitamin B.sub.12 ; 2000 mg     methyl testosterone, and 2000 mg. diethylstilbestrol.                    

                                      TABLE X                                     __________________________________________________________________________    Average Daily Weight Gain and Feed Conversion                                 Efficiency in Pigs from 50 Pounds to about 200                                Pounds Fed Antibiotic A-4696 Hydrochloride or a                               Part of the Daily Diet.                                                                            Weight of                                                                              Weight of                                                                              Average   Feed     %                          A-4696 Hydro-                                                                          No. of                                                                             Pigs at start                                                                          Pigs at Con-                                                                           Daily                                                                              %    Conversion                                                                             Im-                 Feed   Chloride, PPM                                                                          Pigs in                                                                            of Test, clusion of Test,                                                                       Gain Improve-                                                                           Efficiency                                                                             prove-              Ration in Feed  Test LBS. Ave.                                                                              LBS. Ave.                                                                              Pounds                                                                             ment Feed/Lb.                                                                               ment                __________________________________________________________________________    Basal  0        72   50       198.2    1.50 --   3.19     --                  Ration                                                                        Basal                                                                         Ration                                                                        Plus A-4696                                                                   Hydro-                                                                        chloride                                                                             5        72   50       196.4    1.56 --   3.18     --                  "      20       72   50       197.1    1.57 --   3.07*    3.8                 "      50       72   50       206.4     1.67*                                                                             5.7  3.06*    4.9                 __________________________________________________________________________     *Statistically significant (P<.05)                                       

The acute toxicity of A-4696 hydrochloride, determined in mice andexpressed as LD₅₀, is 2,400 mg./kg. when administered intraperitoneally.

The novel antibiotic of this invention is produced by culturing a newlydiscovered strain of an Actinoplanes organism under aerobic conditionsin a suitable culture medium until the culture medium containssubstantial antibiotic activity. The antibiotic can be recovered byemploying various isolation and purification procedures commonly usedand understood in the art. When the antibiotic is to be incorporated infeed-stuff for animal use, a lesser degree of purification is requiredthan that which is necessary if the antibiotic is to be used formedicinal purposes.

The microorganism used according to this invention for the production ofantibiotic A-4696 has been identified as a strain of a species ofActinoplanes of the family Actinoplanaceae. The Actinoplanaceae are anew family of microorganisms of the order Actinomycetales, having beenfirst described by Dr. John N. Couch, Jour. Elisha Mitchell Sci. Soc.,65, 315-318 (1949); and 66, 87-92 (1950); Trans. New York Acad. Sci.,16, 315-318 (1954); Jour. Elisha Mitchell Sci. Soc., 71, 148-155 and 269(1955); Bergey's Manual of Determinative Bacteriology, 7th Edition,825-829 (1957); and Jour. Elisha Mitchell Sci. Soc., 79, 53-70 (1963).

The Actinoplanes sp. useful for the production of antibiotic A-4696 hasbeen deposited and made a part of the stock culture collection of theAmerican Type Culture Collection, Rockville, Maryland, from which it isavailable to the public without restriction under the number ATCC 23342.

The Actinoplanes sp. useful for the production of A4696 was isolatedfrom a sample of soil obtained from the Cascade mountain area in thestate of Washington. This organism has been designated number 581 in thecollection of Dr. John N. Couch at the University of North Carolina.

Actinoplanes sp., strain ATCC 23342, is characterized by the physicaland cultural properties set forth in the following paragraphs. Thesystem of Ridgeway, Color Standards and Nomenclature, (1912), isemployed for the naming of the colors.

Microscopic Morphology and General Cultural Characteristics ofActinoplanes sp. ATCC 23342

Microscopic Morphology - Mycelia are rather sparse on Liquidamber (Sweetgum tree) pollen in water. The hyphae are branched, septate, 0.2-1.5μ.in diameter. Hyphae fill the pollen grain, extending out in the water toa distance about equal to the diameter of the grain. Sporangiophoresproject above the surface of the water; sporangial stalks, usuallysingle, at times branched to bear two, or rarely more, sporangia on thesame sporangiophore; stalks 1.0-1.5μ., thick, septate. Sporangia arerather small, 4-11μ. in diameter, subglobose, rarely globose, usuallywith an irregular wall. Mature spores are arranged in one or moreindistinct coils in the sporangium. Sporangial dehiscence is produced bythe swelling of an intersporal substance which causes the sporangium toenlarge and assume an almost smooth spherical shape. Spores are motile,about 1.0-1.5μ., in diameter; globose to subglobose. CulturalCharacteristics on: Czapek Agar (S. A. Waksman, The Actinomycetes,1950). Growth is good; the point inoculum about 2 cms. in diameter aftereight weeks. The central part is mound-like; the smear flattened andminutely bumpy. The color of the mycelia is a brilliant zinc orange; theagar is pale buff to distinctly yellowish. Sporangia are rarely formed.Peptone Czapek Agar (5 g. of peptone substituted for 2 g. of sodiumnitrite). Growth is similar to that observed on Czapek Agar, butfurrows, ridges and bumps are more distinct than on Czapek Agar. Nosporangia are formed.

As previously noted, Actinoplanes sp., strain ATCC 23342, can be grownin a culture medium to produce antibiotic A-4696. The culture medium canbe any one of a number of different media. However, for economy inproduction, maximum yield, and ease of isolation of the antibioticcertain culture media are preferred. Thus, for example, starch is one ofthe preferred sources of carbohydrate, and soybean meal is one of thepreferred nitrogen sources. Other carbohydrate sources which can be usedinclude molasses, glucose, dextrin, glycerol, and the like. Nitrogensources also include amino acid mixtures, peptones, and the like.

Nutrient inorganic salts to be incorporated in the culture media caninclude the customary salts capable of yielding sodium, potassium,ammonia, calcium, phosphate, chloride, sulfate, acetate, carbohydrate,and like ions. Additionally, sources of growth factors, such asdistillers' solubles and yeast extracts, can be included with beneficialeffect on the production of A-4696 antibiotic. As is necessary for thegrowth and development of other microorganisms, essential trace elementsshould also be included in the culture medium for growing theActinoplanes sp. employed in this invention. Such trace elements arecommonly supplied as impurities incidental to the addition of the otherconstituents of the medium.

The organism used to produce A-4696 can be grown over a relatively widepH range. However, it is desirable to adjust the pH of the culturemedium to between about the pH 6.5 and pH 7.2 before inoculation withthe organism. As with other Actinomycetes, the pH of the growing mediumgradually changes during the growth period; the pH at the end of thefermentation period usually ranging from about 7.0 to 7.8.

Submerged aerobic cultural conditions are preferred for the productionof A-4696. Relatively small amounts of the antibiotic can be produced byshake flask culture; however, for the preparation of large amounts,submerged aerobic culture in sterile tanks is preferred. The culturemedium in the sterile tank can be inoculated with a sporulated ormycelia frament suspension; but because of the time lag experienced whena sporulated suspension is used, the preferred inoculum is thevegetative form of the culture. More efficient use of fermentationequipment is realized by avoiding the time lag in the growth cycle.

Accordingly, it is desirable to produce a vegetative inoculum of theorganism by inoculating a relatively small quantity of culture mediumwith the spores or mycelial fragments of the organism, and when a youngactive vegetative inoculum is obtained aseptically transfer it to thelarge tank. The medium in which the vegetative inoculum is grown can bethe same as that utilized for large-scale fermentation of A-4696,although other media can be employed.

The A-4696 producing Actinoplanes sp., strain ATCC 23342, grows attemperatures between about 20° and 40° C. The largest amounts of A-4696appear to be produced at a temperature of about 30° C.

Sterile air is blown through the culture medium in the submerged aerobicculture process. The volume of air sparged into the culture mediumvaries from about 0.1 to about 1.0 volume of air per minute per volumeof culture medium. The most efficient growth and antibiotic productionare achieved when the volume of air is at least 1/2 volume of air perminute per volume of culture medium.

The rate of production of A-4696 and the concentration of antibioticactivity in the culture medium can be followed during the growth periodby testing samples of the fermentation broth for antibiotic acitivityagainst organisms known to be susceptible to the antibiotic. One suchassay organism useful in the present invention is Bacillus subtilis. Thebioassay can be carried out by the standard turbidimetric or cup-platemethods, or by the paper disc assay on agar plates.

Generally, maximum production of the anitbiotic occurs within about 4 to6 days in shake flasks or submerged aerobic culture fermentations.

Antibiotic A-4696 can be recovered from the culture medium and separatedfrom other substances which may be present by adsorptive and extractivetechniques. Adsorptive techniques are preferred because such proceduresavoid the use of large volumes of solvents required in extractionprocesses.

After fermentation, the antibiotic acitivity is present in both thebroth and mycelium. The culture medium is filtered to separate thefermentation broth from the solid mycelia. The mycelial cake is washedwith water and then slurried in water adjusted to a pH of 10.5 with 5.0NNaOH, agitated vigorously for 30 minutes and filtered. The two filtratesand the wash water are combined and the antibiotic activity adsorbedtherefrom on a suitable adsorptive agent, such as activitated carbon,activated acid alumina, polyamide resin, cellulose, silica gel, and thelike. The adsorption can be accomplished by passing the antibioticactivity-containing solution over a bed of the adsorbent, or by addingthe adsorbent to the solution, mixing thoroughly for 20 to 30 minutesand filtering off the exhausted solvent. The antibiotic adhering to theadsorbent can be recovered as impure A-4696 by customary elutionprocedures.

The crude A-4696 can be purified by the preparation of the picrate saltand the conversion thereof to the hydrochloride salt, or by adsorptionand elution procedures using an appropriate adsorbent or ion exchangeresin. Suitable adsorbents include Polyamide resin (M. Woelm, Eschwege,Germany), acidic alumina, neutral alumina, basic alumina, silica gel,Amberlite XAD-2 (Rohm and Haas, Philadelphia), Amberlite XAD-4 (Rohm andHaas, Philadelphia), and Dowex 50 (H⁺) (Dow Chemical, Midland, Mich.).

This invention is further illustrated by the following examples:

EXAMPLE 5 A. Shake Flask Fermentation of Antibiotic A-4696

Mycelial fragments of Actinoplanes sp., strain ATCC 23342 wereinoculated on a nutrient agar slant having the following composition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Pre-cooked oatmeal   60      g.                                               Yeast                2.5     g.                                               K.sub.2 HPO.sub.4    1.0     g.                                               Dried distiller's solubles                                                                         5.0     g.                                               Czapek's mineral stock*                                                                            5.0     ml.                                              Agar                 25      g.                                               Water, deionized     1       l.                                               *Czapek's mineral stock has the following composition:                        KCl                  100     g.                                               MgSO.sub.4 7H.sub.2 O                                                                              100     g.                                               FeSo.sub.4 7H.sub.2 O                                                                              2       g.                                               (Dissolve in two mls. conc. HCl)                                              Water, deionized     1       l.                                               ______________________________________                                    

The slant was inoculated with ATCC 23342 and incubated for 6 days at 30°C. The culture does not normally sporulate on this medium, and it isnecessary to macerate the mycelial mat with a flattened, sharpened,inoculating needle in order to increase the number of potential growthcenters. The macerated mature culture was covered with sterile distilledwater and scraped carefully with a sterile rod to obtain a mycelialsuspension.

The suspension thus obtained was used to inoculate 100 ml. of a sterilevegetative medium having the following composition:

    ______________________________________                                        Ingredient            Amount                                                  ______________________________________                                        Glucose               5.0     g.                                              Dextrin               20.0    g.                                              Soybean meal          15.0    g.                                              Yeast extract         2.5     g.                                              Calcium carbonate     1.0     g.                                              Water, tap            1       l.                                              ______________________________________                                    

The inoculated vegetative medium was grown for 48 hours at 30° C. on arotary shaker operating at 250 rpm.

Ten ml. of the incubated vegetative medium was inoculated into 100 ml.of a sterile "bump" medium of the same composition as given next above.The thus inoculated "bump" medium was incubated for 24 hours at 30° C.with constant shaking on a rotary shaker operating at 250 rpm.

Four-tenths ml. of the incubated "bump" medium was inoculated into 100ml. portions of a production medium of the composition shown belowcontained in 500 ml. Erlenmeyer flasks, and sterilized at 120° C. for 30minutes:

    ______________________________________                                        Ingredient            Percent                                                 ______________________________________                                        Dextrose              1.0                                                     Dextrin               3.0                                                     Peptone               1.5                                                     Soybean meal          0.5                                                     MgSO.sub.4 7H.sub.2 O 0.2                                                     Molasses, beet sugar  1.5                                                     Corn steep liquor     0.5                                                     Betaine               0.1                                                     K.sub.2 HPO.sub.4     0.05                                                    Water, deionized, q.s.                                                                              25.1.                                                   ______________________________________                                    

The pH of the medium was adjusted to 7.5 with 5N sodium hydroxidesolution before sterilization. After sterilization the pH wasapproximately 6.9.

The production fermentation was shaken for about 96 hours at atemperature of 30° C. on a rotary shaker operating at 250 rpm. The pH atthe end of the fermentation cycle was about 7.2.

B. 40-liter tank fermentation of antibiotic A-4696

The preparation of the inoculum proceeded through the incubation of the"bump" medium detailed under section A, above Twenty-five liters of aproduction medium as outlined above, with 0.02% Dow Corning antifoamadded, was sterilized by autoclaving at 120° C. for 30 minutes andcharged into a 40 l. fermentation tank. One-hundred milliliters ofincubated "bump" medium was inoculated into the sterile productionmedium. The inoculated production medium contained in the 40 l. tank wasallowed to ferment for 4 days at 30° C. The fermentation was aeratedwith sterile air in an amount of about one-half volume of air per volumeof culture medium per minute. The fermenting production medium wasagitated with a mixer utilizing an impeller of a proper size and turningat an appropriate rpm to insure adequate mixing of air with the medium.The pH of the culture medium gradually increased from an initial levelof about 6.9 to about 7.2 as the fermentation proceeded.

C. Isolation of Antibiotic A-4696

The whole broth obtained from an A-4696 fermentation, as describedabove, was filtered with the aid of a commercial filter aid. Thefiltrate was set aside. The mycelial cake was washed with 32 l. of waterand the wash water set aside. The mycelial cake was then suspended in anadditional 32 l. of water and the pH of the mixture adjusted to pH 10.5with 5N sodium hydroxide solution. The mycelial cake water suspensionwas stirred for 45 minutes and the mixture was filtered. This filtrateand the water wash were combined with the original filtrate from thefermentation broth and the pH of combined filtrates was adjusted to pH4.0 with H₂ SO₄. The acidified combined filtrates was passed through acarbon column utilizing 1 kg. of activated carbon, (Pittsburgh, 12 ×40). The activated carbon column was washed until the effluent wascolorless. The A-4696 activity was adsorbed on he carbon column. TheA-4696 activity was eluted from the carbon column utilizing a 1% H₂ SO₄solution in acetone: H₂ O (1:1). Two liters of the acidifiedacetone-water solution was sufficient to elute the A-4696 activity fromthe carbon column. The eluate containing the A-4696 activity was treatedwith a saturated barium hydroxide solution, in order to form aprecipitate of barium sulfate, thus removing the sulfate ions from thesolution.

The mixture was filtered and the barium sulfate precipitate wasdiscarded. The filtrate containing the A-4696 activity was concentratedunder vacuum to dryness. The resulting residue comprising the A-4696activity amounted to approximately 80 g.

D. Conversion of A-4696 Activity to Crude A-4696 Hydrochloride

The approximately 80 g. of A-4696 activity was taken up in a volume of 5l. of water, and then treated with 500 g. of activated carbon (DarcoG-60, Atlas Chemical, Wilmington, Del.) This mixture was stirred for 1hour and then filtered. The filtrate was discarded. The carbon filtercake containing the A-4696 activity was washed with 1 l. of water andthe water wash was discarded. The carbon filter cake was further washedwith 1 l. of 0.05N aqueous hydrochloric acid. The acid wash wasdiscarded. The washed carbon cake was eluted by stirring 30 minutes with500 ml. of an aqueous hydrochloric acidacetone solution (0.05NHCl:acetone[7:3]). The mixture was filtered and the filtrate set aside.The elution of the activated carbon was repeated 4 times in the samemanner, each time setting aside the filtrate. The five eluatescontaining the A-4696 activity were combined. The combined eluates werethen concentrated under vacuum to a volume of approximately 100 ml.Two-hundred milliliters of methanol was added to the aqueous concentratecontaining the A-4696 activity. Two liters of acetone was added to thisaqueousmethanol solution. A precipitate, consisting of crude A-4696hydrochloride, formed in the acetone-aqueous methanol solution. Afterfiltering and drying the precipitate, 60.9 g., of crude A-4696hydrochloride was recovered.

E. Preparation of Crystalline A-4696 Hydrochloride

Twenty-five grams of A-4696 hydrochloride, prepared according to theprocedure outlined in section D of this example, was dissolved in 20 ml.of water. The A-4696 hydrochloride solution was passed over a waterwashed Polyamide resin bed, (M. Woelm, Eschwege, Germany), contained ina glass column of approximately 7 × 60 centimeters. The effluent was setaside. The Polyamide resin column was washed with water at a flow rateof approximately 8-10 ml. per minute. The antimicrobial activity of thecolumn effluent was measured by conventional procedures. The effluentscontaining antimicrobial activity were combined and concentrated todryness under vacuum. The residue was dissolved in a mixture of 25 ml.water and 50 ml. methanol. This aqueous methanol solution of A-4696hydrochloride was acidified to a pH of 2.0 with 5N HCl. Approximately1.5 l. of acetone was added to the aqueous methanol solution toprecipitate the hydrochloride salt therefrom.

The filter cake containing the A-4696 hydrochloride was dissolved inminimum quantity of water. An amount of ethanol equal to twice thevolume of water was then added and the mixture was heated toapproximately 60° C. The mixture was then cooled and the hydrochloridesalt of A-4696 crystallized therefrom. The crystals were filtered anddried. Approximately 9 g. of crystalline A-4696 hydrochloride wasrecovered.

EXAMPLE 6

Purification of A-4696 Over Acid-washed Alumina

Two and three-tenths grams of crude A-4696, obtained as described inExample 1 through section D above, was dissolved in 20 ml. of 50 percentaqueous methanol and run over a 2 × 4 cm. column of acid-washed alumina(Alcoa). The column was immediately washed with methanol. The activitywas eluted from the acid alumina with 50% aqueous methanol. Aliquots ofthe elution were collected and the antimicrobial activity determined oneach fraction. The fractions which exhibited antimicrobial activity werecombined, concentrated to approximately 100 ml. and acetone added toprecipitate the A-4696. The precipitate was filtered and dried in vacuo,yielding approximately 930 mg. of pure crystalline A-4696.

The acid-washed alumina used in the purification described above wasprepared as follows: Alcoa activiated alumina F-20 was washed with wateradjusted to pH 3 with H₂ SO₄ by stirring for 6 hours maintaining the pHat 3.0. The alumina was filtered off, washed with 50% aqueous methanol,and dried in an oven at 100° C.

EXAMPLE 7 Purification of A-4696 by the Preparation of the Picrate Saltand Conversion thereof to the Hydrochloride Salt

Five-hundred milligrams of crude A-4696, obtained as described inExample 1 through section D above, was dissolved in 25 ml. of water and25 ml. of a saturated aqueous solution of picric acid were added withstirring. The mixture was allowed to stand overnight at 5° C. and aprecipitate formed. The precipitate was centrifuged off and dried. Theprecipitate thus obtained was dissolved in 25 ml. methanol, the pH wasadjusted to 1.5 with HCl, and added to 500 ml. of diethyl ether toprecipitate the A-4696 hydrochloride salt. The hydrochloride salt wascentrifuged off, washed with diethyl ether and dried in vacuo yieldingapproximately 305 mg. of A-4696 hydrochloride as a whit crystallinesalt.

EXAMPLE 8 Preparation of A-4696 Sulfate Salt

Two grams of A-4696 hydrochloride, obtained as described in Example 1through section E above, was dissolved in 300 ml. of water, the pHadjusted to 7.5 with 5N sodium hydroxide, and 30 g. of Darco G-60 carbonwas added. The mixture was stirred for 30 minutes; filtered with the aidof a commercial filter aid, and the filter cake washed successively with300 ml. each of water and 0.05N H₂ SO₄.

The activity was eluted from the filter cake by adding the filter caketo 500 ml. of a mixture consisting of 70% 0.05N H₂ SO₄ and 30% acetone,stirring for 30 minutes and filtering to remove the carbon. The filtratewas concentrated to approximately 10 milliliters. About 20 ml. ofmethanol was added to the concentrate and the thus formed solution wasadded to 600 ml. of acetone to precipitate the A-4696 sultate. Theprecipitate was filtered off; washed with acetone and dried in vacuo,yielding 862 mg. of A-4696 sulfate as a white crystalline salt.

What is claimed is:
 1. Factor A of antibiotic A-4696, or apharmaceutically acceptable acid addition salt thereof, which antibioticin the form of its hydrochloride salt is a white, crystalline substancesoluble in water and insoluble in the common organic solvents; has theapproximate composition of 50.21 percent carbon, 5.48 percent hydrogen,4.96 percent nitrogen, 30.42 percent oxygen, and 6.96 percent chlorine;has a specific rotation, [α]_(D) ²⁵ of -81° (c=0.8, H₂ O); has anultraviolet adsorption maximum in acidic and neutral solutions at 282mμ. with an extinction coefficient E_(1cm).^(1%) of 35.9; and has thefollowing distinguishable bands in its infrared absorption spectrum whendetermined in a mineral oil mull: 3.0 broad, 3.44, 4.25, 4.31, 6.07,6.20, 6.31, 6.65, 7.04, 7.73, 8.10, 8.21, 8.47, 8.89, 9.43, 9.69, 9.83,10.14, 11.07, 11.36 and 12.25 microns.
 2. Factor B of antibiotic A-4696,or a pharmaceutically acceptable acid addition salt thereof, whichfactor in the form of its hydrochloride salt is a white, crystallinesubstance soluble in water and insoluble in the common organic solvents;has the approximate composition of 50.12 percent carbon, 4.97 percenthydrogen, 5.51 percent nitrogen, 29.45 percent oxygen, and 6.20 percentchlorine; has a specific rotation, [α]_(D) ²⁵ of -108.3 (c=1, H₂ O); hasan ultraviolet absorption maximum in acidic and neutral solutions at 280mμ. with an extinction coefficient E_(1cm).^(1%) of 51.2; and has thefollowing distinguishable bands in its infrared absorption spectrum whendetermined in a mineral oil mull: 3.0 broad, 3.44, 4.25, 4.31, 6.07,6.20, 6.31, 6.66, 7.04, 7.75, 8.13, 8.24, 8.48, 8.91, 9.43, 9.69, 9.83,10.14, 11.07, 11.36 and 12.25 microns.